1. Field of the Invention
This invention relates to a treating and preventing agent for metabolic bone diseases containing human neurotrophin-3. Moreover, it concerns treating and preventing agent for diseases about ossification especially for bone fractures by using NT-3 as a new osteoblastic proliferative factor.
2. Description of the Prior Art
At the limited part, bone repeats ossification and bone resorption for substituting new bones for old bones to maintain endoskeleton as support function. It is also prepared to respond quickly to the change of various mechanical stresses and mineral balances. This bone reformation is carried out based on the coupling of mainly both bone resorption type cells such as osteoclasts and ossification type cells such as osteoblasts. Recently, it appears that the function of osteoblasts is not only ossification but also playing a role as a control center of cell chain reaction for bone reformation phenomenon that is closely related to the differentiation and activation of osteoclasts (Inoue, T., Mebio (1990), Special Version p.2-7).
In the bone metabolic diseases, there are such as osteoporosis, Paget's disease, osteomalacia, hyperostosis, osteopetrosis and so on. Especially, frequency of osteoporosis is very high, incidentally more than a half population of postmenopausal women and aged, and the diagnosis and effective treatment for it are strongly desired.
Bone metabolic diseases are accompanied with metabolic disorders that are specific to bones at cell level in some bone tissues. It is very effective for elucidation of these metabolic disorders to discover, separate and identify the factor that relates specifically to bone metabolism. The present inventors investigated hard to discover the specific factor of bone metabolism, and finally the present invention was accomplished.
In detail, the present inventors especially used an osteoblastic cell line which functions mainly for calcification and identified the protein produced from that cell line.
NT-3 (neurotrophin-3), presented by the present invention, is a protein discovered by Hohn, A. et al., and Maisonpierre, P. C. et al., and has an effect to promote the nerve growth. NT-3 is a protein which consists of 119 amino acid subunits dimer with Tyr on N-terminal and Thr on C-terminal, and these amino acid sequences are shown in the literature. NT-3 is available at Pepro Tech Inc., USA (Rocky Hill, N.H. 08533, USA, catalogue No. 450-03). The above mentioned literature describes the gene coding NT-3, and NT-3 can be prepared based by the method described on the literature. They were known to be four kinds of factors in the nerve growth factor family including NT-3 by now. These are nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and human neurotrophin-4, other than NT-3. The function of BDNF is to increase the number of AChE positive neuron at culture system of hippocampus, and that of NT-3 is to increase the number of calcium binding protein (Calbinin) positive neuron (Ip, N.Y. et al. J.Neurosci vol.13, P.3394-3405, 1993). Moreover, it is also reported that NT-3 increases the proliferation of precursor cells of neural crest (Kalcheim, C. et al. Proc. Natl. Acad. Sci., USA, vol.89, P.1661-1665, 1992).
On the other hand, the receptors of the NGF family are trk family and trkA, trkB, trkC are known by now (Snider, W. D. Cell vol. 77, p.627-638, 1994). NGF, BDNF and NT-3 are known to bind especially to trkA, trkB and trkC, respectively. Trk is a proto-oncogene which arises from colon cancer and has the tyrosine kinase type receptor structure including the half of C-terminal side tyrosine kinase domain. The amino acid sequence of this tyrosine kinase domain has high homology with other receptor types of tyrosine kinase. On the activity of tyrosine kinase, for example, trkA which is a receptor of NGF responded to NGF, subjected to tyrosine phosphorylation, and shows tyrosine kinase activity. By this tyrosine kinase, MAP2 (microtuble-associated protein 2, Boulton, T. G. et al. Cell vol.65, p.663-675, 1991) and phospholipase C are subjected to tyrosine phosphorylation, and consequently incorporation of calcium ion influx occurs. Proliferation signals are transmitted by this signal transmission form (Ikeuchi, T. et al., Experimental Medicine vol.10, p.126-131, 1992). By this binding of tyrosine kinase type receptor such as trkC and ligands, tyrosine kinase is activated and signal transmission is initiated. Ca.sup.2+ channel activated by binding of ligands and receptors is called "receptor working Ca.sup.2+ channel". It is checked in various cells such as lymphocyte, smooth muscle (Kuno, M., Experimental Medicine, vol.7, p.73-78, 1989).
It has been observed that MC3T3-E1 of the mouse osteoblast like cell line is expressing mRNA of NT-3 and the amount of mRNA can be increased by treating MC3T3-E1 cells with TGF-.beta. (Nakanishi, et al., Biochem. Biophys. Res. Commun. vol.198, p.891-897, 1994). MC3T3-E1 is a cell line established by Kodama, H. et al., which has ability of calcification and was derived from new born mouse calvaria (Jpn. J. OralBiol. Vol. 23, p.899-901, 1981, appeared in General Catalog of RIKEN GENE BANK, No.1 April, 1995). However, no reports have been presented so far that NT-3 works as an osteoblast proliferative factor.